Research in the area of T cell biology has exploded in the last 10 years, and the field continues to expand, as the various roles of T cells in various biological processes become understood, or better understood.
It has now become accepted that T cells play a key role in tumor immunology. Specifically, T cells recognize various complexes of MHC Class I molecules and peptides on the surfaces of cells, and lyse these "targets". The processes by which this occurs are complex, and are discussed in many well known publications.
Decreased activity of T cells is seen in tumor patients, and it has been suggested that depression of T cell activity leads to circumvention of immunological control of tumors. Several immunotherapeutic approaches have been developed, with the common aim of restoring T cell activity in the patient. See, e.g., Pardoll, Immunol. Today 14: 310-316 (1993), Beun et al., Immunol. Today 15: 11-15 (1994), both of which are incorporated by reference, for a general overview of the field.
One of the more successful strategies which has been developed is the use of bispecific monoclonal antibodies ("Bi-MAbs" hereafter), which target and activate a broad range of resting T cells against antigen positive tumor cells, thereby inducing tumor cell destruction. See Renner & Pfreundschuh, Immunol. Rev. 145: 179-191 (1995), incorporated by reference. In a particular application of this technique, Hodgkin's lymphoma was used as a model. Combining two Bi-MAbs, the first of which consisted of one arm which recognizes Hodgkin's associated CD30 antigen, the second of which recognizes either the CD3 trigger or the CD28 trigger molecule, led to efficient tumor cell lysis, both in vitro and in vivo. See Renner et al., Science 264: 833-835 (1994), incorporated by reference. As reported by Renner et al., Blood 87: 2930-2937 (1996), SCID mice bearing Hodgkin's lymphoma responded favorably to this approach.
Additional studies on the mechanisms involved in Bi-MAb mediated T cell activation and tumor cell lysis, revealed that multiple activation markers, cytokines, and cytokine proteins, are upregulated rapidly after combined CD3 and CD28 antigen crosslinking. See Renner et al., Eur. J. Immunol. 25: 2027-2032 (1995). The effectiveness of this approach is based on the fact that Bi-MAb mediated T cell stimulation mimics the physiological pathways of T cell activation, which depend on two signals. As a rule, one signal is delivered by activation of the T cell receptor, via a peptide presented in an MHC restricted fashion, and the second, distinct signal is provided via the interaction of the CD28 antigen on T cells, and a member of the B-7 family on the corresponding "APC", or antigen presenting cell. See Linsley et al., J. Exp. Med. 173: 721-730 (1991); Linsley et al., Ann. Rev. Immunol. 11: 191-212 (1993).
Expression of genes in cells is frequently related to the effect of various molecules on cells. Study of the changes in expression of various genes requires that one have various analytical models available for deployment. One such method is differential display, or differential mRNA display. This method is taught by Liang et al., Science 257: 967-971 (1992); Liang et al., Curr. Opin. Immunol. 7: 274-280 (1995), both of which are incorporated by reference, as well as in U.S. Pat. No. 5,762,311 to Pardee and Liang, also incorporated by reference. Briefly, performance of this methodology requires the isolation of total RNA from two cell populations which are to be compared. Following isolation, first strand copies of both RNAs are made by reverse transcription, using oligo-dT primers having specific dinucleotides at their end. This is followed by a polymerase chain reaction, wherein this 3'-primer and an arbitrary 5'-primer are used for the generation of cDNA fragments. The use of the short, arbitrary primers frees the investigator from the need to depend on known DNA sequences, and makes it much easier to search for unknown genes.
Investigations have been carried out wherein differential display methodologies have been used to isolate genes which are differentially expressed in malignancies (e.g. brain tumors), heart disease and diabetes. See Zhang et al, Mol. Carcinog. 8: 123-126 (1993), Utans et al., Proc. Natl. Acad. Sci. U.S.A. 91: 6463-6467 (1994); Joseph et al., Biochem. Biophys. Res. Comm. 20: 1227-1234 (1994). This approach has also been used for breast cancer studies, and several cDNA fragments were characterized which are upregulated in tumor cells, as compared to normal tissues. The role of these genes in tumorigenesis remains to be elucidated. See Liang et al., Cancer Res. 52: 6966-6968 (1992).
The present application involves the application of the differential display technique to T cell activation, and the identification of molecules involved in this process. Specifically, the application is directed to the identification of those molecules which are differentially expressed following combined stimulation by CD3 and CD28 trigger molecules. This will be clear from the disclosure which follows.